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WO1992019721A1 - Expression genique dans des bacilles - Google Patents

Expression genique dans des bacilles Download PDF

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Publication number
WO1992019721A1
WO1992019721A1 PCT/US1992/003403 US9203403W WO9219721A1 WO 1992019721 A1 WO1992019721 A1 WO 1992019721A1 US 9203403 W US9203403 W US 9203403W WO 9219721 A1 WO9219721 A1 WO 9219721A1
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WO
WIPO (PCT)
Prior art keywords
enzyme
bacillus
promoter
gene
regulatory genes
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Application number
PCT/US1992/003403
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English (en)
Inventor
Eugenio Ferrari
Original Assignee
Genencor International, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genencor International, Inc. filed Critical Genencor International, Inc.
Priority to KR1019930703241A priority Critical patent/KR100230613B1/ko
Priority to CA002108358A priority patent/CA2108358C/fr
Priority to FI934717A priority patent/FI934717A0/fi
Priority to DE69230825T priority patent/DE69230825T2/de
Priority to EP92911495A priority patent/EP0600893B1/fr
Priority to AU19111/92A priority patent/AU663321B2/en
Priority to JP4511425A priority patent/JP2654472B2/ja
Priority to DK92911495T priority patent/DK0600893T3/da
Publication of WO1992019721A1 publication Critical patent/WO1992019721A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus

Definitions

  • the invention relates to novel genes, vectors and Bacilli transformed with them useful in the hyperproduction of enzymes. Specifically, the invention relates to specific combinations of unlinked genes which, either mutated or wild type, synergistically induce hyperproduction of enzymes.
  • Bacillus subtilis 1-168 is the most extensively studied member of the Bacilli and has a well developed genetic system as the result of many years of academic research. As a result, there is a considerable amount of knowledge pertaining to the expression of extracellular enzymes and many mutants of B. subtilis 1-168 exist that are altered in the expression of these enzymes. There are several different known genes which code for proteins that regulate the expression of extracellular enzymes. Although the exact molecular mechanism of this regulation is not completely
  • Regions of the ENA around promoters can actively interact with certain transcriptional control factors and effectively regulate the strength and efficiency of a particular promoter. This interaction will result either in a stimulation or in a repression of the transcription of that particular target gene. As a consequence, mutations in either the genes coding for these transcriptional control factors or in regions of the chromosome with which they interact are expected to yield more or less mRNA of the target gene. This in turn will results in higher or lower amount of enzyme synthesized by the cell.
  • sacU genes which have the most pleiotropic effect among all these known regulatory genes.
  • a strain carrying for example sacU(Hy) mutations can sporulate in presence of high levels of glucose, has a very low efficiency of transformation, lacks flagella and it is also responsible for the hyperprcducticn of several secreted enzymes. (Dedonder et al, Microbiology 1976, see above).
  • the sacQ gene from three different bacilli has been cloned and
  • the gene isolated from B. subtilis encodes a 46 amino acid polypeptide.
  • the sacQ gene when hyperexpressed either because of a mutation in its promoter or when present in a multicopy plasmid causes hyper production of certain extracellular enzymes, including the alkaline protease (Yang et al, 1986, J. Bacteriol 166, 113-119).
  • the prtR gene encodes a 60 amino acid polypeptide. When this gene is present in B. subtilis in a multicopy plasmid it causes increased
  • the hpr gene encodes a 203 amino acid protein and certain mutations in this gene stimulate alkaline protease production. It has been established that mutations which cause the loss of this gene product stimulates transcription of the subtilisin gene (Perego and Hoch, 1988, J. Bacteriol. 170, 2560-2567). The product of the hpr gene therefore is most likely a repressor of the subtilisin gene. It has also been determined that the scoC gene and possibly the catA gene are identical to the hpr. The notation hpr will be used thereafter to refer to any of the three alleles called hpr, scoC or catA.
  • the sin gene encodes a polypeptide of 111 amino acids (Gaur et al, 1986, J. Bacteriol, 168:860-869).
  • An insertional ina ⁇ tivation of the gene causes the production of a higher level of alkaline protease, suggesting that this gene also acts as a repressor of the expression of alkaline protease.
  • abrB gene codes for a polypeptide of 97 amino acids. Also for this gene there is evidence that it is a repressor of the transcription of the alkaline protease and might have an important role in the temporal regulation of this gene.
  • the invention relates to a Bacillus able to hyperprcduce an enzyme comprising:
  • mutated genes are preferably selected from the group comprising:
  • the invention also relates to vectors comprising the novel gene, to cells-transformed with the vectors and to a processing for making enzymes.
  • Figure 1 shows the construction of the pSAR gene.
  • Figure 2 shows the construction of the pSAICM gene.
  • Figure 3 shows the integration of the pSAICM gene into the B. subtilis chromosome.
  • Applicants have demonstrated novel cxambination of mutated or wild type genes, vectors and Bacilli transformed with them which are unique in their ability to induce hyperproduction of enzymes.
  • Two mutated genes as described above are introduced in a strain carrying an appropriate promoter and gene sequence.
  • the promoter sequence and the gene sequence of the enzyme are introduced into such host strain by mean of an integrative vector.
  • Such vector which may or may not be amplified upon integration, is stably maintained as part of the host genome and replicates within the cell as integral part of its chromosome, integration can occur at different region of the chromosome including but not limited to the region of homology with the promoter used in a particular construction.
  • Such host may have its original enzyme sequences, or other gene sequences,
  • enzyme gene sequence refers to a ENA sequence encoding for the expression of an enzyme, in general either heterologous or homologous to Bacilli.
  • enzymes are known and within the scope of this teaching including but not limited to those preferred enzymes from
  • Bacillus lentus Bacillus amyloliquefaciens (e.g., ATCC 23844), Bacillus licheniformis (e.g., ATCC 21415), Bacillus alcal ⁇ philus (e.g., ATCC 21522), B. subtilis (e.g., ATCC 6051) and Bacillus cereus (e.g., ATCC 21768). Also included are all the genes capable of being expressed in Bacillus such as the ones coding for proteases, lipases, cutinases, esterases,
  • subtilisin A preferred enzyme is subtilisin.
  • Other preferred enzymes include endoglycosidase H from Streptomyces species and lipase from Pseud ⁇ monas mendocino ATCC 53552 (available from American Type Culture Collection, Rockville, MD). Enzymes such as subtilisin which act at an optimum at higher pH are among the preferred enzymes. It is also desirable to act at an optimum at higher pH. It is also
  • the enzyme gene sequence also includes the DNA sequence encoding for a naturally occurring enzyme gene sequence vSiich is modified to produce a mutant ENA sequence which encodes a substitution, insertion or deletion of one or more amino acids in the gene sequence. Suitable modification methods are disclosed in, for example, EPO Publication No. 0130756, published January 9, 1985.
  • Subtilisins are bacterial alkaline proteases which generally act to cleave peptide bonds of proteins or peptides.
  • subtilisin means a naturally occurring subtilisin or a reccmbinant subtilisin.
  • a series of naturally occurring subtilisins is known to be produced and often secreted by various bacterial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity.
  • This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases.
  • subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine.
  • subtilisin related proteases the relative order of these animo acids, reading from the amino to carboxyl terminus is
  • subtilisin herein refers to a serine protease.
  • Recombinant subtilisin refers to a subtilisin in which the DNA sequence encoding the subtilisin is modified to produce a mutant ENA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally occurring subtilisin amino acid sequence. Suitable methods to produce such modification include those disclosed herein and in EPO Publication No. 0130756.
  • a subtilisin multiple mutant containing the substitution of methionine at amino acid residues 50, 124 and 222 with phenylalanine, isoleucine and glutamine, respectively can be considered to be derived from the rec ⁇ mbinant subtilisin containing the substitution of glutamine at residue 222 (Gln-222) disclosed in EPO
  • the multiple mutant thus is produced by the substitution of phenylalanine for methionine at residue 50 and isoleucine for methionine at residue 124 in the Gln-222 re ⁇ ombinant subtilisin.
  • a functional promoter sequence controlling the expression of the enzyme gene operably linked to the enzyme gene sequence refers to a promoter sequence which, controls the transcription and translation of the coding sequence in Bacillus.
  • the promoter sequence is chosen so that it is functional in the microorganism, chosen for expression.
  • promoter sequence including ribosomal binding sites formed from Lambda P (Renaut, et al., Gene 15, 81 [1981]) as well as the trp (Russell, et al., Gene 20, 23 (1982)or tac (de Boer et al., PNAS. USA 80, 21 [1983]) may be cperably linked to coding sequences to express alkaline protease in E.
  • B. subtilis 1-168 is the expression host, the B ⁇ . subtilis alkaline protease promoter (Stahl et al, J. Bacteriol 158, 411-418 [1984]) alpha amylase promoter of B. subtilis (Yang et al., 1983, Nucleic Acids Res. 11, 237-249) or B. amyloliquefaciens (Tarkinen, et al, J. Biol. Chem. 258, 1007-1013 [1983]), the neutral protease promoter from B. subtilis
  • transcriptional regulatory genes refers to any of the genes whose product affects the level of transcription from the enzyme promoter. These genes product can interact at the promoter level (e.g. AbrB) or with a region upstream or downstream of it (e.g. Hpr, SacU, SacQ, PrtR, Sin) either directly or indirectly.
  • the definition above is comprehensive of the gene product or the lacik of it and any possible mutation which may increase the level of transcription of a particular promoter (e.g. multiple copies). This definition is meant to include but not limited to certain mutations in the promoter region, downstream or upstream of the promoter region of the target gene which may result in an increase in transcription due to a better interaction or lack of it with the regulatory genes or with the transcriptional machinery (e.g. RNA polymerase, mutations which decrease production of a gene product which interferes with transcription). These genes and the effect of their mutations are described above. These specific combinations, applicant has discovered, exhibit a synergistic effect when compared with the results predicted from either single mutations alone.
  • “Expression vector” refers to a ENA construct containing a ENA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the ENA in a suitable host.
  • control sequences can include a promoter to effect transcription or optional operation sequence to control such transcription, a sequence encoding a suitable ribosome binding sites (RBS) and sequences which control
  • the vector may be a plasmid, a phage particle, or a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host gene or may integrate into the gene itself.
  • a preferred vector is pSAI.
  • Bacillus host cells for the purpose of this invention are those
  • Bacillus hosts capable of functionally accepting the expression vector Preferably, they have been manipulated by the methods disclosed in EPO Publication No. 0130756 to render them incapable of secreting other enzymes deleterious to the desired enzyme.
  • a preferred host cell for expressing subtilisin is the B. subtilis strain BG 2036 which is deficient in
  • subtilisin enzymatically active neutral protease and alkaline protease
  • the construction of strain BG 2036 is described in detail in EPO Publication No. 0130756 and further described by Yang, et al (1984) J. Bacteriol. 16015-21.
  • Other host cells for expressing subtilisin include any derivative of B. subtilis 1-168 and any other member of the bacillus family in which such system might work (i.e. B. amyloliquefaciens and B. licheniformis (EPO Publication No. 0130756).
  • operably linked describes the relationship between two ENA regions and means they are functionally related to one another.
  • a promoter is operably linked to the gene sequence coding for an alkaline protease if it properly controls the transcription of the sequence.
  • subtilisin gene from B. amyloliquefaciens was isolated as described by Wells et al, 1983, Nucleic Acid Res. 11: 7911-7925. Briefly, partially Sau3A digested chromosomal ENA from B. amyloliquefaciens was ligated to BamHl digested pBS42 and used to transform E. coli.
  • pBS42 is a plasmid vector composed of an origin of replication from pUB110 recognized as such by B. subtilis, an origin of replication from pBR322 which allows
  • acetyltransferase from pC194, a fully characterized plasmid from Staphilococcus aureus (Horinouchi and Wei ⁇ blum, 1982), which can express CmR in both E. coli and B. subtilis.
  • Synthetic ENA probes designed from the known amino acid sequence of the subtilisin were used to isolate a plasmid (p54) carrying the ENA coding for the gene.
  • the isolated ENA fragment was able to code for subtilisin production when transferred to B. subtilis and the ENA sequence translated to match the published amino acid sequence of the alkaline protease (Wells et al, 1983, Nucleic Acid Res. 11: 7911-7925) .
  • a Sau3A BamH1 fragment from pS4-5 (a derivative of pS4) comprising the subtilisin structural gene from the 8th codon of the signal sequence to 250 bp after the 3' end of the gene (all of which is B. amyloliquefaciens ENA), was fused to a 750 bp EcoR1-Sau3A fragment which carries the promoter and the first 8 codons of the translated sequence of the B. subtilis subtilisin gene (Fig. 1).
  • B. subtilis consists of 1.6 Kb from pC194 and the subtilisin gene joined together by short linker regions.
  • One linker is composed of 20 bp from pUC9, and the other one consists of the 17 bp from pBR322 included between the EcoR1 and the Cla1 sites.
  • chromosomal mutations known to increase the level of secreted enzymes either through competent cell transformation or PBS1 transduction. Both these techniques are commonly used for genetic studies in B. subtilis.
  • the resulting strain is a prototrophic sporeforming derivative of B. subtilis 1-168 (catalog No. 1- A1, Bacillus Genetic Stock Center) which carries the B. amyloliquefaciens subtilisin gene and the CmR gene from pC194 integrated into the chromosome.
  • chloramphenicol resistance The resulting chloramphenicol resistant strains were scored on solid medium (tryptose blood agar base agar, TBAB) containing skim milk for the detection of the hyperprotease producing characteristic (due to the presence of the scoC, catA and hpr mutations) of the parent strain.
  • PBS-1 lysates (see above) were prepared on the ScoC, CatA and Hpr strains carrying the pSAI integrative plasmid by a standard published technique (Hcch et al 1967, see above). These lysates were used to transduce strain BG2097.
  • transductant Same of the transductant were screened en plates containing 1.6% skim milk and compared to BG3002. Transductants able to yield a larger halo on these type of plates were selected and strains BG 3008, BG3005 and BG3007 were obtained.
  • the genotype of these strains is isogeneic (hisA, aprE, nprE, pSAI::CmR) except for the hpr gene were the mutations scoCl, catA or hpr?7 are present respectively.
  • representatives of this transduction were examined in shake flasks using a nutrient broth medium, they produced -19-17mg/L subtilisin or 5-10 fold more than the strain that did not contain the hyperproducing mutation.
  • SacU(Hy) strains One of the properties of SacU(Hy) strains is that they lack the ability to produce flagella and to became motile. Since PBS-1 attaches to motile flagella, it is impossible to prepare a PBS-1 working lysate on SacU(By) strains. This problem was circumvented by the isolation of a spontaneous mutant that was motile and retained its protease hyperproducing phenotype. The nature of the mutation that results in motility is unknown. A lysate was prepared on the motile SacU(Hy) strain.
  • This lysate was used to transduce either BG3002 ( ⁇ apr, ⁇ npr, hisA) containing the pSAI plasmid to histidine prototropy. Since the sacU gene and the hisA gene are linked by transduction, a certain
  • strains containing both the sacU(Hy)32 and either the scoC or catA mutations exhibited a larger zone of clearing on skim milk plates than did either of the strains containing the individual mutations. These strains produced ⁇ 55mg/L in nutrient broth medium.
  • SacQ(By) strains A lysate was prepared on a SacQ(Hy) strain. This lysate was used to transduce a strain to threonine
  • sacQ(Hy) and either the scoC or catA mutation could be constructed by using a combination of the above procedures.
  • the catA/scoC mutation was introduced in one step along with the plasmid pSAI, followed by the introduction of the saoQ(Hy) mutation and finally the sacU(Hy) mutation was introduced into the strain.

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Abstract

Cette invention concerne des gènes, des vecteurs et des bacilles transformés avec ces derniers qui sont utiles dans l'hyperproduction d'enzymes, ainsi que des procédés permettant de produire des enzymes en utilisant les gènes, les vecteurs et les organismes de cette invention.
PCT/US1992/003403 1991-04-26 1992-04-24 Expression genique dans des bacilles WO1992019721A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
KR1019930703241A KR100230613B1 (ko) 1991-04-26 1992-04-24 간균내 유전자 발현
CA002108358A CA2108358C (fr) 1991-04-26 1992-04-24 Expression de genes dans des bacilles
FI934717A FI934717A0 (fi) 1991-04-26 1992-04-24 Genexpression i bacillus
DE69230825T DE69230825T2 (de) 1991-04-26 1992-04-24 Genexpression in bazillen
EP92911495A EP0600893B1 (fr) 1991-04-26 1992-04-24 Expression genique dans des bacilles
AU19111/92A AU663321B2 (en) 1991-04-26 1992-04-24 Gene expression in bacilli
JP4511425A JP2654472B2 (ja) 1991-04-26 1992-04-24 バシラス属中の遺伝子発現
DK92911495T DK0600893T3 (da) 1991-04-26 1992-04-24 Genekspression i Bacilli

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US69210891A 1991-04-26 1991-04-26
US692,108 1991-04-26

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WO1992019721A1 true WO1992019721A1 (fr) 1992-11-12

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PCT/US1992/003403 WO1992019721A1 (fr) 1991-04-26 1992-04-24 Expression genique dans des bacilles

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US (1) US5387521A (fr)
EP (1) EP0600893B1 (fr)
JP (1) JP2654472B2 (fr)
KR (1) KR100230613B1 (fr)
AU (1) AU663321B2 (fr)
CA (1) CA2108358C (fr)
DE (1) DE69230825T2 (fr)
DK (1) DK0600893T3 (fr)
FI (1) FI934717A0 (fr)
NZ (1) NZ242507A (fr)
WO (1) WO1992019721A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993020214A1 (fr) * 1992-03-30 1993-10-14 Genencor International, Inc. Expression genique heterologue dans le bacillum subtilis: approche par fusion
WO2003083125A1 (fr) * 2002-03-29 2003-10-09 Genencor International, Inc. Expression proteinique amelioree dans bacillus

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5646044A (en) * 1995-03-02 1997-07-08 Henkel Kommanditgesellschaft Auf Aktien Expression systems for the production of target proteins in bacillus
GB9828711D0 (en) * 1998-12-24 1999-02-17 Genencor Int Production of proteins in gram-positive microorganisms
EP1472347B1 (fr) * 2002-02-15 2011-01-12 Danisco US Inc. Accroissement de l expression de proteines dans le bacillus subtilis
US20050026189A1 (en) * 2003-05-29 2005-02-03 Liangsu Wang Microbial operons
US20090048136A1 (en) * 2007-08-15 2009-02-19 Mcdonald Hugh C Kappa-carrageenase and kappa-carrageenase-containing compositions
AR076941A1 (es) 2009-06-11 2011-07-20 Danisco Us Inc Cepa de bacillus para una mayor produccion de proteina

Citations (1)

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Publication number Priority date Publication date Assignee Title
US4801541A (en) * 1981-09-11 1989-01-31 The University Of Rochester Method of increasing the yield of a product by altering a microorganism

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
FR2582316B1 (fr) * 1985-05-22 1988-12-02 Centre Nat Rech Scient Vecteurs d'expression de l'a-amylase dans bacillus subtilis, souches obtenues et procede de preparation d'a-amylase

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US4801541A (en) * 1981-09-11 1989-01-31 The University Of Rochester Method of increasing the yield of a product by altering a microorganism

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Applied and Environmental Microbiology, Volume 39, No. 1, issued January 1980, YONEDA et al., "Increased Production of Extracellular Enzymes by the Synergistic Effect of Genes Introduced into Bacillus subtilis by Stepwise Transformation", pages 274-276, see entire document. *
Gene, Volume 83, issued 1989, WONG, "Development of an inducible and enhanciboe expression and secretion system in Bacillus subtilis", pages 215-223. *
Journal of Bacteriology, Volume 170, No. 1, issued January 1988, HENNER et al., "Location of the Targets of the hpr-97, sacO32(Hy), and sacU36(Hy) Mutations in Upstream Regions of the Subtilisin Promoter", pages 296-300, see entire document, especially the introduction. *
See also references of EP0600893A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993020214A1 (fr) * 1992-03-30 1993-10-14 Genencor International, Inc. Expression genique heterologue dans le bacillum subtilis: approche par fusion
WO2003083125A1 (fr) * 2002-03-29 2003-10-09 Genencor International, Inc. Expression proteinique amelioree dans bacillus
US8124399B2 (en) 2002-03-29 2012-02-28 Danisco Us Inc. Enhanced protein expression in Bacillus
US8383366B2 (en) 2002-03-29 2013-02-26 Danisco Us Inc. Enhanced protein expression in Bacillus
US9175294B2 (en) 2002-03-29 2015-11-03 Danisco Us Inc. Enhanced protein expression in Bacillus

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JPH06507075A (ja) 1994-08-11
JP2654472B2 (ja) 1997-09-17
DE69230825T2 (de) 2000-11-30
NZ242507A (en) 1994-04-27
DE69230825D1 (de) 2000-04-27
US5387521A (en) 1995-02-07
EP0600893B1 (fr) 2000-03-22
AU1911192A (en) 1992-12-21
CA2108358A1 (fr) 1992-10-27
FI934717L (fi) 1993-10-25
KR100230613B1 (ko) 1999-12-01
AU663321B2 (en) 1995-10-05
EP0600893A4 (fr) 1994-11-30
CA2108358C (fr) 2002-11-19
EP0600893A1 (fr) 1994-06-15
DK0600893T3 (da) 2000-08-21
FI934717A0 (fi) 1993-10-25

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